For years, electrophoresis has been used to separate and isolate macromolecules and particles due to differences in apparent surface charge. When two substances have the same charge but different mobilities, the faster moving substances will overrun and migrate ahead of the slower one. This is usually due to substance geometry and other hydrodynamic considerations, e.g., macromolecular group conformation/surface topography, etc. In general, the affinity of one substance for another will affect the mobility of that substance when both are subjected to electrophoresis. It is thus theoretically possible to maximize separation of particles with the same or similar charge through the use of specific affinity compounds. For a general review of affinity electrophoresis (of macromolecules), see Takeo, Electrophoresis 5:187-195 (1984).
Affinity electrophoresis techniques are of enormous potential value in a variety of medical biochemical areas (e.g., research, diagnostics). Of particular importance are techniques for isolating or separating very large molecules or cells such as cancer cells, blood cells, DNA molecules, lymphocytes, proteins, etc. New techniques are needed since in a great number of cases, the cells or particles of interest are not amenable to presently known electrophoretic separation techniques. This is particularly true for cells which are easily damaged by the extreme conditions such as temperature, pH, salt concentration or fluid shear inherent in various electrophoretic techniques. It is also true that most biological cells have reasonably similar electrophoretic mobilities (approximately 0.9 .+-.0.3 .mu.ms.sup.-1 V.sup.-1 cm), making them hard to separate electrophoretically. Electrophoretic particle separation and analysis systems need to be specific for various types of particles and cells. It is extremely important that the range of electrophoretic mobility be entirely controlled such as by the addition of compounds which vary mobility in a specific manner, and, preferably in a concentration-dependent manner.
The use of affinity agents such as antibodies to enhance the specificity of electrophoresis and develop a useful system by which a large number of particles and cells could be separated was theorized as early as the 1920's. However, antibody-induced cell aggregation, and a demonstrated lack of cell-antibody interaction appreciably altering substance electrophoretic mobility has kept this goal at arms length. Previous research in this field attempted to use immuno-compounds and other agents to affect electrophoretic mobility of various cells. For example, Seaman, pgs. 1135-1229 in The Red Blood Cell, Vol. 3 (1975), discloses modifying red blood cell (erythrocyte) surfaces with such materials as antisera, enzymes, and neutral polymers. In the first two cases (antisera and enzymes), problems were reported including aggregation, limited reduction of cell mobility, and a general lack of specificity in the treatments. It was found that the use of freely absorbed neutral polymers actually increased particle electrophoretic mobility. Effects of unmodified antibodies on red blood cells (such as indicated above) has also been reported by Sachtleben, in Cell Electrophoresis, pgs. 100-114 (1965). The use of a double antibody system yielding up to about a 40% reduction in electrophoretic mobility in epithelial cells and lymphocytes was reported in Cohly et al in Cell Electrophoresis, pgs. 611-616 (1985). In an attempt to alter net cell surface charge, rather than cell surface electrohydrodynamics, these authors exposed cells covered with antibodies to secondary anti-antibodies possessing a net positive charge which they hoped would partially neutralize the cells native net negative charge (due primarily to surface carboxylate groups). These previous attempts in reducing mobility generally suffered from various problems such as aggregation, and large, controlled reductions in electrophoretic mobility have not been achieved.
In the patent art, many attempts at separating biological cells and other compounds through electrophoresis are known. For instance, U.S. Pat. No. 4,474,886 (Willard) relates to the use of SDS polyacrylamide gel in gel electrophoresis wherein the SDS gel binds to proteins to aid in separation by mass. The SDS is charged and uniformly coats the macromolecule creating uniform charge per unit area. Thus separation is on the basis of surface area which is related to size. SDS kills cells, which regardless cannot be subjected to such gel electrophoresis. Independent of these blocks, this method would not aid in separating two molecular samples of similar mass which have different charges.
As indicated above, the use of neutral polymers in affecting electrophoretic mobility has been examined. It has been observed that when freely absorbed, such polymers typically cause an increase, and not a decrease, in the electrophoretic mobility (see Brooks, J. Colloid Interf. Sci., 1971 and Seaman, above). Various researchers have employed charged polymers in methods to obtain decreases in the mobility of macromolecules but not particles. Examples of these techniques are described in references such as Shimura et al., Electrophoresis 8:135-139 (1987) (charged polylysine added to ligand to aid biomolecule separation), and Vestermark, German Patent No. 2,040,091 (electrophoretic separation achieved through use of charged polyethylene glycol ions). These techniques do not aid in "masking" the native, i.e., effective surface charge of macromolecules, particles or cells, because the polymers in these cases are of opposite charge and will neutralize, not remove or mask, native charges on the macromolecules. What has not been previously recognized or accomplished, therefore, is the development of a system by which hydrophilic, uncharged polymers, such as polyethylene glycol (PEG), can be used to substantially reduce electrophoretic mobility by utilization of their polymer segment viscosity properties to alter the hydrodynamic nature of the "surface" of cells, particles and macromolecules in a concentration-dependent manner and/or in a manner based on their specific antigenic characteristics.
It is thus highly desirable to develop a system which will be able to employ polyethylene glycol or other hydrophilic neutral polymer in an affinity compound and which can retard electrophoretic mobility in a concentration-dependent manner so as to allow separation of a greater variety of cells, particles, macromolecules, or other substances.